Nocardia aciditolerans sp. nov., isolated from a spruce forest soil

Actinomycetes growing on acidified starch-casein agar seeded with suspensions of litter and mineral soil from a spruce forest were provisionally assigned to the genus Nocardia based upon colonial properties. Representative isolates were found to grow optimally at pH 5.5, have chemotaxonomic and morphological features consistent with their assignment to the genus Nocardia and formed two closely related subclades in the Nocardia 16S rRNA gene tree. DNA:DNA relatedness assays showed that representatives of the subclades belong to a single genomic species. The isolates were distantly associated with their nearest phylogenetic neighbour, the type strain of Nocardia kruczakiae, and were distinguished readily from the latter based on phenotypic properties. On the basis of these data it is proposed that the isolates merit recognition as a new species, Nocardia aciditolerans sp. nov. The type strain is isolate CSCA68^T (=KACC 17155^T = NCIMB 14829^T = DSM 45801^T).     

Streptacidiphilus durhamensis sp. nov., isolated from a spruce forest soil

The taxonomic position of three acidophilic actinobacteria, strains FGG38, FGG39 and FSCA67^T, isolated from the fermentation litter layer of a spruce forest soil was established using a polyphasic approach. The strains were shown to have chemotaxonomic and morphological properties consistent with their classification in the genus Streptacidiphilus and formed a distinct phyletic line in the Streptacidiphilus 16S rRNA gene tree being most closely related to Streptacidiphilus albus DSM 41753^T (99.4 % similarity). DNA:DNA relatedness data showed that isolate FSCA67^T and the type strain of S. albus belonged to markedly distinct genomic species. The isolates had many phenotypic properties in common and were distinguished readily from their closest phylogenetic neighbours in the Streptacidiphilus gene tree using a broad range of these features. Based on the combined genotypic and phenotypic data the three isolates are considered to represent a new Streptacidiphilus species. The name Streptacidiphilus durhamensis sp. nov. is proposed for this taxon with isolate FSCA67^T (=DSM 45796^T = KACC 17154^T = NCIMB 14829^T) as the type strain.     

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world''s most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials ⁹, vaccines ^{14, 15}, electronic materials, chemical tools, and molecular electronic containers^{4, 5, 10, 11, 16, 18, 12}.In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups ⁸. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications ⁸. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed ¹³. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus ³, Carnation mottle virus ¹², Cowpea chlorotic mottle virus ⁶, Tobacco mosaic virus ¹⁷, Turnip yellow mosaic virus ¹, and MRFV ¹³.     

Streptomyces erringtonii sp. nov. and Streptomyces kaempferi sp. nov., isolated from a hay meadow soil

Two filamentous actinomycetes isolated from a hay meadow soil were provisionally assigned to the genus Streptomyces based on morphological features. The isolates were found to have chemical and morphological properties typical of the genus Streptomyces and formed distinct phyletic lines in the 16S rRNA gene tree. Isolate I36^T was most closely related to Streptomyces glauciniger NBRC 100913^T and isolate I37^T to Streptomyces mirabilis NBRC 13450^T. Low DNA:DNA relatedness values were recorded between each of the isolates and their closest phylogenetic neighbour. The isolates were also distinguished from their nearest phylogenetic neighbour, and from one another, using a combination of phenotypic properties. These data indicate that the isolates should be recognised as new species in the genus Streptomyces. The names proposed for these new taxa are Streptomyces erringtonii sp. nov. and Streptomyces kaempferi sp. nov. with isolate I36^T (=CGMCC 4.7016^T = KACC 15424^T) and isolate I37^T (=CGMCC 4.7020^T = KACC 15428^T) as the respective type strains.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: I. IDENTIFICATION IN tRNA FROM ETIOLATED PHASEOLUS VULGARIS L. SEEDLINGS

The cytokinin-active ribonucleosides present in tRNA from etiolated Phaseolus vulgaris L. seedlings have been isolated and identified as cis-ribosylzeatin, 2-methylthio-ribosylzeatin, and N⁶-(Δ²-isopentenyl)-adenosine. The structures of the compounds were established on the basis of their chromatographic properties and the mass spectra of their permethylated and perdeuteromethylated derivatives. Cis-ribosylzeatin was the major cytokinin-active constituent of tRNA from this source.     

Excision Repair Properties of Isogenic rec− Mutants of Escherichia coli K-12

We have examined the excision repair properties of isogenic rec⁻ and uvr⁻ strains of Escherichia coli K-12. A recB⁻recC⁻ strain excises dimers at a rate nearly that of the rec⁺ parent, reaching the same extent of excision after a 1-hr postirradiation incubation. recA⁻ and recA⁻recB⁻ strains excise 75 to 80% of the dimers excised by their rec⁺ parent, whereas a uvrB⁻ strain excises no dimers during a 1-hr incubation. The doses of ultraviolet light (254 nm) required to reduce survival to 37% of the original population are 8 ergs/mm² for recA or recA recB mutants, 5 ergs/mm² for the uvrB⁻ strain, 30 ergs/mm² for the recB recC mutant, and 230 ergs/mm² for the wild-type parent. From these data one cannot account for the ultraviolet light sensitivity of rec⁻ strains on the basis of their excision repair properties. We conclude that rec gene products play no significant role in the early steps of excision repair. The assay we have used for excision of thymine dimers is a modification of the Carrier-Setlow technique, and is described in detail in the Appendix to this paper. To show the properties and validity of this method, results of experiments with thymine dimers formed in vitro and in vivo in E. coli K-12 are presented. These results show our method to be reproducible and sensitive to 0.005% of the total radioactive thymine present in thymine-containing dimers.     

Comparison of three RE agents—99mTc-phytate, 99mTc-sulfur colloid and 99mTc-Sb2S3 colloid in the rodent species

^99mTc-phytate, ^99mTc-sulfur colloid and ^99mTc-antimony sulfide colloid are recognised as RE agents in clinical nuclear medicine. Since these three agents exist in vitro in different forms of particulate aggregation it was of interest to compare their properties and to evaluate the merits and limitations of each of these agents. A comparison was carried out in rats and rabbits as a prelude to a systematic study in human subjects. Quality tested radiopharmaceuticals were prepared and their pharmacokinetics studied in rats. The blood clearance, tissue distribution and scinti-imaging patterns of the RE system were also compared in rabbits. All the agents have excellent localizing properties in the liver but differ in varying extents in their disposition in other tissues. ^99mTc-sulfur colloid has some predisposition for the lungs while ^99mTc-phytate has a little propensity for the kidney and gut at late time periods. ^99mTc-Sb₂S₃ colloid shows a more pronounced concentration in the bone-marrow, and displays intermediate properties between ^99mTc-phytate and ^99mTc-sulfur colloid, besides being the most stable (in vitro) radiopharmaceutical in this series of RE agents.     

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca²⁺-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K_d = ∼0.2 μm), and one is on CBD2 (K_d = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K_d values but differ dramatically in their Ca²⁺ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca²⁺ ions (k_r = 280 s⁻¹, k_f = 7 s⁻¹, and k_s = 0.4 s⁻¹), whereas the dissociation of two Ca²⁺ ions from CBD1 (k_f = 16 s⁻¹) and one Ca²⁺ ion from CBD2 (k_r = 125 s⁻¹) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca²⁺, whereas the kinetic and equilibrium properties of three Ca²⁺ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca²⁺ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca²⁺ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.     

Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I^tb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I^tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I^tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.     

The Matricellular Protein Periostin Is Required for Sost Inhibition and the Anabolic Response to Mechanical Loading and Physical Activity

Periostin (gene Postn) is a secreted extracellular matrix protein involved in cell recruitment and adhesion and plays an important role in odontogenesis. In bone, periostin is preferentially expressed in the periosteum, but its functional significance remains unclear. We investigated Postn^−/− mice and their wild type littermates to elucidate the role of periostin in the skeletal response to moderate physical activity and direct axial compression of the tibia. Furthermore, we administered a sclerostin-blocking antibody to these mice in order to demonstrate the influence of sustained Sost expression in their altered bone phenotypes. Cancellous and cortical bone microarchitecture as well as bending strength were altered in Postn^−/− compared with Postn^+/+ mice. Exercise and axial compression both significantly increased bone mineral density and trabecular and cortical microarchitecture as well as biomechanical properties of the long bones in Postn^+/+ mice by increasing the bone formation activity, particularly at the periosteum. These changes correlated with an increase of periostin expression and a consecutive decrease of Sost in the stimulated bones. In contrast, mechanical stimuli had no effect on the skeletal properties of Postn^−/− mice, where base-line expression of Sost levels were higher than Postn^+/+ and remained unchanged following axial compression. In turn, the concomitant injection of sclerostin-blocking antibody rescued the bone biomechanical response in Postn^−/− mice. Taken together, these results indicate that the matricellular periostin protein is required for Sost inhibition and thereby plays an important role in the determination of bone mass and microstructural in response to loading.     

Ca2+ signaling of human pluripotent stem cells-derived cardiomyocytes as compared to adult mammalian cardiomyocytes

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) have been extensively used for in vitro modeling of human cardiovascular disease, drug screening and pharmacotherapy, but little rigorous studies have been reported on their biophysical or Ca²⁺ signaling properties. There is also considerable concern as to the level of their maturity and whether they can serve as reliable models for adult human cardiac myocytes. Ultrastructural difference such as lack of t-tubular network, their polygonal shapes, disorganized sarcomeric myofilament, and their rhythmic automaticity, among others, have been cited as evidence for immaturity of hiPSC-CMs. In this review, we will deal with Ca²⁺ signaling, its regulation, and its stage of maturity as compared to the mammalian adult cardiomyocytes. We shall summarize the data on functional aspects of Ca²⁺signaling and its parameters that include: L-type calcium channel (Cav1.2), I_Ca-induced Ca²⁺release, CICR, and its parameters, cardiac Na/Ca exchanger (NCX1), the ryanodine receptors (RyR2), sarco-reticular Ca²⁺pump, SERCA2a/PLB, and the contribution of mitochondrial Ca²⁺ to hiPSC-CMs excitation-contraction (EC)-coupling as compared with adult mammalian cardiomyocytes. The comparative studies suggest that qualitatively hiPSC-CMs have similar Ca²⁺signaling properties as those of adult cardiomyocytes, but quantitative differences do exist. This review, we hope, will allow the readers to judge for themselves to what extent Ca²⁺signaling of hiPSC-CMs represents the adult form of this signaling pathway, and whether these cells can be used as good models of human cardiomyocytes.     

Skin extracellular matrix components accelerate the regenerative potential of Lin− cells

Due to their unique properties, bone marrow-derived Lin^? cells can be used to regenerate damaged tissues, including skin. The objective of our study was to determine the influence of the skin tissue-specific microenvironment on mouse Lin^? cell proliferation and migration in vitro. Cells were analyzed for the expression of stem/progenitor surface markers by flow cytometry. Proliferation of MACS-purified cells in 3D cultures was investigated by WST-8 assay. Lin^? cell migration was evaluated by in vitro scratch assay. The results obtained show that basement membrane matrix is more effective for Lin^? cell proliferation in vitro. However, type I collagen matrix better enhances the re-epithelization process, that depends on the cell migratory properties. These studies are important for preparing cells to be used in transplantation.     

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNA^Phe and tRNA_init, Escherichia coli tRNA_init and HIV tRNA^Lys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem. 2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg²⁺. Results showed a stabilizing effect associated with the presence of the modifications and Mg²⁺ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNA_init and limitations were observed in the conformation of E. coli tRNA_init, the presence of the modified nucleotides and of Mg²⁺ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.     

Gemini Alkyldeoxy-D-Glucitolammonium Salts as Modern Surfactants and Microbiocides: Synthesis,Antimicrobial and Surface Activity,Biodegradation

Dimeric quaternary alkylammonium salts possess a favourable surface and antimicrobial activity. In this paper we describe synthesis, spectroscopic analysis, surface and antimicrobial activity as well as biodegradability of polymethylene-α,ω-bis(N,N-dialkyl-N-deoxy-D-glucitolammonium iodides), a new group of dimeric quaternary ammonium salts. This new group of gemini surfactants can be produced from chemicals which come from renewable sources. The structure of products has been determined by the FTIR and ¹H and ¹³C NMR spectroscopy. The biodegradability, surface activity and antimicrobial efficacy against Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus niger and Penicillium chrysogenum were determined. The influence of the number of alkyl chains and their lengths on surface and antimicrobial properties has been shown. In general, dimeric quaternary alkyldeoxy-D-glucitolammonium salts with long alkyl substituents show favourable surface properties and an excellent antimicrobial activity.     

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: I. Mannosylated units

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[¹⁴C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [¹⁴C]Man material specifically soluble in CHCl₃/CH₃OH/H₂O, 10 : 10 : 3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some $\text{dolichol}\text{-P-[}{}^{14}\text{C}\text{]}\text{Man}$). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to α-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N′-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [¹⁴C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.     

Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA⁺ and rodA⁻ strains. Tapping-mode AFM images of wild-type and rodA⁺ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA⁻ spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA⁺ spores. Rodlets were removed from rodA⁺ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA⁺ and rodA⁻ spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Salvia suspension cultures as production systems for oleanolic and ursolic acid

Oleanolic acid (OA) and ursolic acid (UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from Salvia officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the respiration activity monitoring system (RAMOS^®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The µ_max values for these suspension cultures ranged from 0.20 to 0.37 day^?1, their OA and UA contents were greater than 1.3 and 1.2 mg g^?1, respectively, and they afforded maximum volumetric yields of 21.0 mg l^?1 for OA and 32.8 mg l^?1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.     

Influence of anticonvulsant cannabinoids on posttetanic potentiation at isolated bullfrog ganglia

The effects of anticonvulsant cannabinoids on posttetanic potentiation (PTP) at bullfrog paravertebral ganglia $\text{in vitro}$ were investigated electrophysiologically. Two Δ⁹-tetrahydrocannabinol metabolites, 11-hydroxy-Δ⁹-tetrahydrocannabinol and 8α, 11-dihydroxy-Δ⁹-tetrahydrocannabinol, as well as cannabidiol, markedly depressed PTP. In contrast, Δ⁹-tetrahydrocannabinol had no such effect. Thus, the findings of the present study clearly demonstrate that pharmacological properties of these hydroxylated metabolites of Δ⁹-tetrahydrocannabinol are not identical to those of their parent compound.     

Dynamics of voltage profile in enzymatic ion transporters, demonstrated in electrokinetics of proton pumping rhodopsin

H⁺-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H⁺-pumping rhodopsin. The electrical properties of H⁺-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H⁺-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H⁺ transfer to an H⁺ acceptor, reprotonation of retinal from the intracellular face via an H⁺ donor, and proton release to the extracellular space via an H⁺ release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby—as in an Ohmic voltage divider—the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H⁺-binding sites involved.